Project Summary ? Kim Lentiviruses including HIV-1, HIV-2 and SIV replicate in both activated CD4+ T cells and terminally- differentiated/non-dividing myeloid cells (e.g. macrophages). While HIV-1 rapidly replicates in activated CD4+ T cells, HIV-1 replication in macrophages is kinetically suppressed. Our previous studies found that macrophages harbor an extremely low dNTP concentration (20-40 nM), which kinetically restricts viral reverse transcription, and later that the host SAMHD1 dNTPase is responsible for the limited dNTP level in macrophages, which restricts HIV-1 replication. However, SIVsm and HIV-2 efficiently replicate even in macrophages due to its viral protein X (Vpx) that proteosomally degrades SAMHD1 and then elevates cellular dNTP levels in macrophages. Our long-term premise is that the extremely limited cellular dNTP pool in nondividing myeloid cells creates unique biochemical and virological features of HIV-1 replication, which directly influence viral replication kinetics, genomic diversity, evolution, and ultimately, pathogenesis of HIV-1. Our previously funded research revealed that HIV-1 reverse transcriptase (RT) uniquely displays efficient DNA synthesis capability even at the low macrophage dNTP concentrations, which enables HIV-1 to overcome the SAMHD1-mediated limited dNTPs in macrophages. However, we found that RTs from many SIV and HIV-2 strains exhibit significantly reduced DNA synthesis capability at the low macrophage dNTP concentrations, compared to HIV-1 RTs, supporting that HIV-2/SIV RTs did not evolve to be highly efficient, possibly because Vpx of these viruses elevates dNTP concentrations for their RTs in macrophages. Based on these findings, first, we hypothesize that the RT enzyme kinetics can counteract SAMHD1-mediated limited dNTP pools in the absence of Vpx. This hypothesis predicts that RT of a SIV mutant with Vpx deletion should evolve to be more efficient in DNA synthesis during the in vivo replication in animals in order to overcome the SAMHD1- mediated low dNTP pools in macrophages (as HIV-1 does). Second, ancestral non-primate lentiviruses such as FIV, BIV and EIAV also efficiently replicate in macrophages, and these ancestral lentiviruses do not encode Vpx. Here we will test whether these non-primate lentiviruses counteract their own host SAMHD1 proteins by proteosomally degrading SAMHD1 (as HIV-2/SIV do) or 2) evolving to harbor enzymatically efficient RTs (as HIV-1 does). Third, we reported that HIV-1 frequently incorporates highly abundant non-canonical/mutagenic ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, specifically in macrophages due to the limited canonical dNTP substrates. Since the incorporation of rNTPs is the most abundant DNA damages in cells and is also sequence-specific, we hypothesize that there are rNTP incorporation hot spots throughout HIV-1 genomic sequences, which become mutational hot spots and ultimately enhance HIV-1 mutagenesis in macrophages. Overall, this application aims at gaining knowledge on unique HIV-1 replication mechanism in myeloid cells that serve as long-living viral reservoirs, ultimately developing myeloid specific anti-HIV-1 agents.